Effect of LY294002 and EGF on mitochondrial membrane potential, cleaved caspase-3 status, and morphology after 24 hr at differing oxygen concentrations. ED77 cells exposed to 1%, 5%, or 21% oxygen for 24 hr with and without EGF or LY294002. (A) Dose response for LY294002 after 48 hr incubation. Hatched shaded bars represent cells with intact ψm (live cells); darkly shaded bars represent cells with dissipated ψm (dead cells). Minimum n = 6. Error bars are ± SEM. *p < 0.05 versus NT (intact); +p < 0.05 versus NT (dissipated). (B) Representative raw data plot for 1% O2 24 hr incubation ± EGF or LY. NT, no treatment; EGF, EGF 10 ng/ml; LY, LY294002 40 µM; E+L, EGF 10 ng/ml plus LY294002 40 µM. (C,D,E) Cells were incubated for 24 hr under the oxygen tensions indicated. (C) ***p < 0.001 versus LY (intact); **p < 0.01 versus LY (intact); + + +p < 0.001 versus LY (dissipated); + +p < 0.01 versus LY (dissipated). (D) Difference in all test groups did not reach statistical significance. (E) *p < 0.05 versus LY (intact). (F) First trimester trophoblast cells were exposed to 1%, 5%, and 21% oxygen for 24 hr. Cell lysates were prepared in RIPA and 50 µg protein were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to the cleaved, active form of caspase-3 and caspase-9. Similar results were obtained in two separate experiments. NT, no treatment; EGF, EGF 10 ng/ml; LY, LY294002 40 µM; E+L, EGF 10 ng/ml plus LY294002 40 µM. (G) Cells were loaded with DiOC6, exposed to the 1% O2 for 24 hr then stained again with propidium iodide and were placed on chamber slides and visualized using Nikon fluorescence microscope. (I) NT, no treatment. (II) EGF, EGF 10 ng/ml. (III) LY, LY294002 40 µM. (IV) E+L = EGF 10 ng/ml plus LY294002 40 µM. Original magnifications ×100. (V) Individual live trophoblast. (VI) Trophoblast undergoing apoptosis. Original magnifications ×400.